Lung Collagen Composition and SynthesisIn the lung, collagen is found associated with bronchi, with...

THE JouR~.-AL OF BIOLOGICAL Crimrsm~ Vol. 219, No. 9,Issue of May 10, PP. 2674-2683, 1974

Printed in U.S.A.

Lung Collagen Composition and Synthesis

CHARACTERIZATIOS AKD CHAKGES WITH AGE

KATHRY-~ H. BRADLEY, SALLY D. NCCOXNELL, AND RONALD G. CRYSTAL

From the Section ox Puhm~ary Biochmistry, Natio,lal Heart and Lzmg Institute, Bethesda, AIau&nd 20014

(Received for publication, July 24, 1973)

SUMMARY

In the lung, collagen is found associated with bronchi, with blood vessels, and with the alveolar interstitium; it is known to be fundamental in the maintenance of lung structure and function. Two collagen chains, crl and cy*, can be extracted from rabbit lung homogenates; each chain has an approximate molecular weight of 100,000 and is composed of amino acids that are characteristic of collagen, but are not specific for lung. Lung slices incubated in vitro synthesize o(, and (Ye chains, as demonstrated by carboxymethylcellulose chromatography, by sodium dodecyl sulfate acrylamide gel electrophoresis, by acidic acrylamide gel electrophoresis, by sensitivity to collagenase, and by sensitivity to trichloroacetic acid at 90”.

As a percentage of dry weight, lung protein does not change significantly during maturation, but the content of collagen increases .5-fold from the late stages of gestation to adult life. The average rate of collagen synthesis per cell decreases slowly IO-fold from fetal to adult life whereas the average rate of noncollagen protein synthesis per cell rapidly decreases to adult levels before birth. The rate of collagen synthesis relative to the rate of total protein synthesis is low in fetal life, rises from 2 to 15% in the first month of life, and then declines by 2 months of age to the fetal level throughout adult life. The maturing lung, therefore, has significant changes in genetic expression during a period of rapid growth as the lung converts to a gas-exchanging organ.

Although lung has no known function during fetal life, it as- sumes a vital role at birth. Whereas morphological development continues beyond birth, by the late stages of gestation, the lung has all of its essential components (1, 2). Fundamental to

this development is the presence of connective tissue elements

which play a central role in lung morphology and mechanical

properties (1, 2).

Lung connective tissue is composed of collagen, elastin, and

ground substance (3-11). Of these, collagen has the greatest

tensile strength and probably plays an important role in limiting

expansion (II). In the lung, collagen is found associated with

bronchi, blood vessels, and the alveolar interstitium (6, 9, 10).

Human lung collagen is composed of (at least) two chains, al and

cyz, made up of amino acids t,hat are characteristic of collagen but

are not specific for lung (12).

This study demonstrates several aspects of rabbit lung col-

lagen including (a) the composition of lung collagen; (b) the

establishment of assays to study in vitro collagen synthesis in

lung slices; and (c) a comparison of the rates of collagen synthe-

sis and noncollagen synthesis during rabbit lung development.

The result of this comparison suggests a change in genetic ex-

pression in the developing lung, as the relative rate of collagen

synthesis is increased and then decreased during maturation.

EXPERIRIESTAL PROCEDURE

Jrlaterials-New Zealand White rabbits, all from an inbred strain, were obtained from B and H Rabbitry, Rockville, Md. Dulbecco’s modified Eagle’s medium [as originally described or with methionine, cystine, or tryptophan omitted] was prepared by the Media Unit, ljivision of Research Services, National In- stit,utes of Health, as was phosphate-buffered saline solution (137 m&f NaCl, 2.68 rnM KCl, s.1 r& Na2HPOI, 1.47 mnl KH,PO,; pH 7.4). Cellulose nitrate filters (type HA, 0.45 pm pore size, 25 mm diameter) were from the Milliporc Corporation. [5-3H]- Proline (31,000 Ci per mole), [2-3H]glycine (33,000 Ci per mole), L-f’%luroline (260 Ci aer mole). nL-hvdroxv-13.4-14ClDroline (54.5 C<pe;-mole), and nL-i5-3B]tryptophan (25$O’Ci pi; mole) were from Schwarz-Mann. L-[35S]Methionine (133,000 Ci per mole) and L-[%]cystine (1700 Ci per mole) were from Amersham-Searle. Liquid scintillators used were: Aquasol (New England Nuclear), Bray’s scintillator (13), Triton X-loo-based scintillator (14), RPI scintillator ((Research Products International, 4 g of 2,5-diphen- yloxazole (PPO) and 50 mg of 1,4-bis[2-(5.phenyloxazolyl)]benzene (POPOP) per 42 ml); RPI-toluene is 42 ml of RPI scintillator per liter of toluene. Collagenase (Form III) was from Advanced Biofactures, Lynbrook, New York.

Eztraction of Collage,1 from Rabbit Lufrg-Four g-week-old rabbits were fed conventional rabbit feed containing 0.3y0 &aminopropionitrile for 10 weeks. then killed. After exsanguination, the iungs were removed and trimmed to remove hi& structures. The trimmed lungs were cut into small pieces and homogenized in 500 ml of 0.5 M acetic acid in a Sorvall Omni-Mixer (16,000 rpm, 2 min). The volume was brought up to 1 liter with 0.5 M acetic acid and the collagen was extracted for 2 days at 4” with stirring. The residue after centrifugation (10,000 X g, 30 min) was re-extracted with 0.5 M acetic acid for 2 days and the resulting residue was again pelleted. The supernatants from the extractions were combined and made to 5y0 NaCl; the collagen was precipitated at 4” for 18 hours with stirring. The precipitated collagen was pelleted (31,000 X g, 30 min), solubilized with 0.5 M acetic acid, clarified (31,ooO X g, 20 min), and reprecipitated in the same way. The partially purified rabbit lung collagen was lyophilized and stored at -20” as a gray filamentcus material (70 mg total).

Collagen was also isolated from 30 I-dav-old rabbits delivered from does maintained on conventional feei. The trimmed lungs were handled as described above, except that only one NaCl pre-

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cipitation was used. The yield of dry, partially purified collagen was 270 mg.

Isolation and Characterization of Collagen ~1 and 012 Chains from Rabbit Lulzg-Seventy milligrams of the partially purified collagen isolated from the lungs of adult lathyritic rabbits were separated into 01 and p components by CM-cellulose chromatography (15). The (~1, 012, pl1, and PI? components were identified by parallel electrophoresis with known standards of pig skin and rabbit skin collagen components on sodium dodecyl sulfate polyacrylamide (5%) gels (16-18) and on acidic polyacrylamide gels (19).

The partially purified collagen isolated from the lungs of newborn rabbits was handled in the same manner.

Extraction of Lung No?lcollagen Proteins-Four newborn rabbits, two 2-month-old rabbits, and one 3-year-old rabbit were exsanguinated and their lungs removed. The lungs of each group were trimmed to remove hilar structures as described above and then homogenized in distilled H20. Two milliliters of each pooled homogenate were digested with collagenase (250 units of collagenase, 20 mM Tris-HCl, pH 7.4, 2.5 mM N-ethylmaleimide, 1 mM CaC12) in a total volume of 2.5 ml for 2 hours at 37” (20). After incubation, 2.5 ml of 20% trichloroacetic acid were added and the homogenates were heated to 90” for 30 min to solubilize any residual collagen (21-23). The remaining precipitate was pelleted (570 X g, 5 min) and the trichloroacetic acid extraction was re- peated at 90”. The resulting precipitate was pelleted as above and washed twice with 57, trichloroacetic acid; the trichloroacetic acid was removed by two ether extractions, and the resulting noncollagen lung protein was dried.

In Vitro Synthesis of Lung Proteins-Lungs were removed from exsanguinated rabbits, the hilar vessels and trachea were excised, and the lungs were washed in phosphate-buffered saline solution at 4” before slicing. The outer 1 mm of lung was discarded (including the pleura) and 0.5.mm slices were made with a Stadie- Riggs slicer (A. H. Thomas) and rinsed in phosphate-buffered saline solution. Rabbits of 20 and 25 days of gestational age (subsequently referred to as fetuses of -10 days and -5 days), as well as rabbits aged 1, 5, 7, and 14 days; 1, 2, 5, and 6 months; and 1, 2, and 3 years were used for these studies. A total of 300 rabbits was used.

Approximately 250 mg (wet weight) of lung slices were rinsed in incubation medium (1 volume of Dulbecco’s modified Eagle’s medium (24) to 1 volume of phosphate-buffered saline solution, 0.5 mM ascorbic acid, 0.6 mM &aminopropionitrile) which had been equilibrated previously with 95% 02, 5% COZ. p-Aminopropio- nitrile was used to inhibit collagen cross-linking (25, 26) and ascorbic acid was used as a cofactor for proline hydroxylation (27, 28). Dulbecco’s modified Eagle’s medium contains no proline, making it suitable for isotopic studies with this amino acid. The rinsed slices were then placed in 1 ml of fresh incubation medium in flat-bottomed glass vials. After 45 min of incubation (37”, 90 oscillations per min in a Dubnoff shaker with a 95% 02, 5% COZ gas phase), the incubation medium was aspirated and discarded, and fresh incubation medium containing [5-3H]proline, [‘*C]proline, [%]cystine, or [%]methionine was added as indicated in the figure and table legends. When [%]methionine or [36S]cystine was used these amino acids were omitted from the medium (respectively). Incubation was then continued for 20 min to 5 hours, as indicated.

Zdentijcalion of in Vitro Product-After incubation, the slices were removed with forceps, placed immediately on a vacuum filtering apparatus with a stainless steel screen (Millipore Cor- poration), and rinsed three times with 10 ml of phosphate-buffered saline solution (4”) with gentle vacuum. The slices were then homogenized in 5 ml of 0.5 M acetic acid for 30 s at 10,000 rpm (Microtip, Polytron homogenizer, Brinkmann Instruments). The homogenate was stirred (15 hours, 4’), clarified by centrifugation (10,000 X g, 10 min), dialyzed against 4 liters of 0.5 M acetic acid at 4” for 24 hours (with 2 changes), then precipitated with 5yo NaCl for 4 hours at 4”. The precipitated collagen was pelleted (31,000 X g, 30 min) and solubilized in 5 ml of 0.04 M sodium acetate, pH 4.8. This material was then dialyzed against 4 liters (with two changes) of column starting buffer at 4’ for 24 hours before chromatography on CM-cellulose as described above and in Fig. 1. Before denaturation, 10 mg of acid-extracted carrier collagen were added (rabbit lung, pig skin, or rabbit skin). After CM-cellulose chromatography, l-ml aliquots (from IO-ml fractions) were counted directly in Aquasol with 30% efficiency for

t

FRACTION NUMBER

FIG. 1. Carboxymethylcellulose chromatography of extracted rabbit lung (Ye and 012 chains. A, partially purified collagen (70 mg), extracted from lungs of 4-month-old rabbits maintained for 10 weeks on P-aminopropionitrile, was solubilized in 12 ml of column starting buffer (0.04 M sodium acetate, pH 4.8), denatured (50”, 15 min), and clarified by centrifugation (1700 g, 5 min) and applied to a CM-cellulose column (1.5 X 10 cm). Collagen chains were eluted with an 800-ml linear NaCl gradient (0 to 0.1 M) at 250 ml per hour and lo-ml fractions were collected. Absorbance at 230 nm (-) is shown. Fractions were pooled and used for amino acid analysis as follows: cy1 (Fractions 20 to 28), PI1 (Frac- tions 33 to 35), &Z (Fractions 42 to 47), ala (Fractions 55 to 60). Fractions between LYE and p11 and between filt and O(Z were shown on sodium dodecyl sulfate acrylamide gels and by amino acid analysis to be a mixture of the two adjacent components. B, [3H]proline-labeled collagen synthesized in 2 hours by 300 mg (wet weight) of lung slices obtained from a a-week-old rabbit was chromatographed on an identical CM-cellulose column under the same conditions. Counts per min per fraction (O---O) and absorbance at. 230 nm (--) are shown. The carrier collagen in this chromatogram was obtained from the skin of 4-month-old lathyritic rabbits.

3H and 80% efficiency for 36S or K?. Alternatively, l-ml fractions were precipitated with 10% trichloroacetic acid at 4” for 10 min, and the precipitates were collected on cellulose nitrate filters, dried, and counted in RPI-toluene with the same efficiencies. Indicated fractions containing [3H]al or [3H](rF ch&ins were pooled, dialyzed against HzO, lyophilized, and solubilized in HaO.

The identity of the [3H]~l and [aH]~ua chains was further verified on sodium dodecyl sulfate polyacrylamide (5yo) gels (16-18) and on acidic polyacrylamide gels (19).

Sensitivity to purified bacterial collagenase was tested by in- cubating lOa cpm of f3H]al or [~H](Y~ chains with collagenase (10 units of collagenase, 20 mM Tris-HCl, pH 7.4, 1 mM CaCll, 2.5 mM N-ethylmaleimide) in a lOO-~1 reaction mixture for 1 hour at 37” (12, 21). Two milliliters of 10% trichloroacetic acid were added to each incubation mixture (10 min, 4”); the precipitates were collected on cellulose nitrate filters and washed with 5% trichloroacetic acid. The filters were dissolved in Bray’s scintillator and counted with 30% efficiency. The sensitivity of the [3H]~l and [3H]o12 chains to collagenase was compared with the sensitivity of [“Hltryptophan-labeled noncollagen protein from lung (20).

Sensitivity to trichloroacetic acid at 90” (12, 21-23) was tested by incubation of 10’ cpm of [3H]proline-labeled 011, [3H]proline- labeled 012, [3H]leucine-labeled rabbit reticulocyte globin, or [3H]- tryptophan-labeled noncollagen protein from rabbit lung in 10%


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trichloroacetic acid at 90” for 30 min, followed by 10 min at 4”. Precipitates wcrc collected on cellrdosc nitrate filters, and washed with 10 ml of 5% trichloroac,ctic acid. The filters were then dissolved in Bray’s scitttillat.or and c.ounted as above.

Comparison of Lu~cg Composition njtrl Rdes of l’rotei,t Synthesis tlztri,cg De1~elop),re,/l-All studies on the rates of protein synthesis wrre done with [W]proline. After incr1bation as described above, slices were removed with forceps and placed immediately on a suction apparatus with a fine steel screen and rinsed t,hree times with 10 ml of phosphate-buffered saline solution (4“) wit,h gentle suction. The slices were then placed in a tube (16 X 150 mm), 2 ml of 0.5 M acetic acid were added, and the slices were homogenized for 30 s at 10,000 rpm (Microtip, I’olytron homogenizer, Brink- mann Instrument,s). A drop of octnnoic acid was added as a de- foaming agent, the homogenate was transferred to a 5.ml gradu- ated cylinder (calibrated, A. II. Thomas), and the volume was made to 3 ml with 0.5 M acetic acid. Aliquots were transferred to individual tubes for s\tbseqllent assays and frozen on Dry ICC. It was fo1und that, fresh homogenates prepared in this manner were easily transferred wit,h disposable micropipettes (Clay Adams), but. if they were frozen in bulk, srtbscqttrnt nccluntc transferring proccdurcs were dillic1tlt brcartse of cl~un~pittg. Aliqtlots were taken as follows: 0.5 ml for dry weight ; 0.5 ml for hydroxyprolinc; 1 ml for I)NA, protein, and specific activity of the isot,ope in the tissrte; and 0.05 ml for labclcd non~oll:~gcn protein.

The dry weight aliqrrots wcrc added to tared tllbcs, lyophilizcd to remove the acetic acid, and reweighed. This method was rc- producible to 0.05 tng.

Assam for H!/drozyproZi,te-Total hydroxvprotinc and [‘“Cl- hydroxyprolinc wcrc determined 011 0.5-1111 :&quot,s of t,issue ho- tnogcnntc by the method of Juva and l’rorkop (29). Optimal results were obtained by incltGon of Norit, A (Fisher; 75 to 100 mg per ml hydrolysate) aft,cr hydrolysis and by chromatographing the second t,olncnc layer on s1licic acid columns t,o remove any residual [‘%]proline t,hat might have been extract,cd wit,h the tolucnc. Each column was washed with tolucne, rcsltlting in a total volume of 20 tnl collected in comnting vials. A 5.ml aliquot was used for the calorimetric assay and 0.65 ml of RPI scintillator wns added to the remaining 15 tnl anti the cpm dctcrrnined wit.h an cfliciency of SOo/o. Aliquots of unl:rbclcd lung hotnogenatcs had known quantit.ies of 114C]hydrox~l~roline added in order to express the [14C]hydroxyprolinc determinations in each sample as 100% recovery.

AssaIls for D.l’A, Profeiu, aud SpwiJic Activiiy of Isotope irr Il’ism--Olle milliliter of homogenate was lyophiliecd t,o remove the acetic acid. To the dry homogenate, 1.0 ml of 10% trichloroacetic acid was added and the tube was mixed to break up the pellet. The protein and nucleic acids were pelleted (1700 X g, 5 min) and used for the l)NA and protein assays. The supcrnatant was decanted to a separate t;tbc and the-precipitate was washed with 1.0 tnl of 5% trichloroacctic acid. The remaining protein and nucleic acids &re pcllcted (1700 X y, 5 min) and the supcrnatant was cornbincd with the previous srtperttatattt.

Speci$c Aclivit?/ of Isofope i/r ‘I’isslle---The cotnbincd supernatant fractions containing the trichloroncctic acid-soluble proline (including [‘%]prolinc) from the tissllc slices were usrd to dct,crmine the specific activity of [“Clproline in the tissue. Any residual precipitate in the sttpernatant fractions was removed by filtering through cellulose nitrate filters; the filtrate was collect,ed. Fifty tnicrolit,ers of the filtrate were added to 10 ml of Aquasol t,o deter- mine the soluble [“Clprolinc in the tissue. Of the filtrate, 1.4 ml were adjusted t,o pH 1 to 7 with 1 N NaOH made to 2 ml with H1O, and u&d to 1ncAsure the total free proline by a modification of the method of Troll and I,ittdsle\ (30).

Assay for D!\‘A a?ld I’rolei,c--‘Ilk pellet, from the trichloroacet.ic acid precipitation (see above) was dissolved in 1.0 1nl of 0.3 N KOII at 37”. The resulting solution was then dilntcd to 5.0 ml with Il*O; a 2.0-ml aliquot was analyzrd for DNA (31) and 0.1 ml was analyzed for protein by the method of Lowry (32) using bovine serum albumin as a standard.

Assay for iVo,ccollagerl I’rolei//-The [14C]proline-labclcd 110~ collagen protein was determined in 50.~1 nliquots of the homog- cnat,c. Two tnillilitcrs of 10% trichloroacetic acid were added to each tube and the collagen &as partially hydrolyzed by heating to 90” for 30 min (21-23). Aft,cr the tubes were cooled to 4” for 20 min, the noncollngcn protein precipitate was pcllctcd (1200 X y, 5 min). The supernatant was decanted and the precipitate was

suspended in 1.0 ml of 5% trichloroacetic acid. After 10 min at 4’ and mixing, the precipitate was collected on a cellulose nitrate filter using 6 ml of 5’;/0 trichloroacetic acid to wash the precipitate. The cellulose nitrate filters wcrc dried and counted in IIPI-toluene with an efficiency of 80%.

CalcuZalio7ts-Several factors arc important, in the calculation of rates of collagen and noncollagcn synthesis in lung tissue slices.

1. Synthesis must be proport,ional to the time for the interval during which the rate is being measured.

2. In tissue slice experiments, there may be a delay in the incorporation of lahcled amino acid into protein since it takes a finite t,ime for the labeled amino acid to move intracellularly. Thus, a plot of incorporation of isotope against titne might not go through the origin, necessitating t.he measurement of incorporation into protein at more than one time point in order to calculate the rate of incorporation (33).

3. The calculat,ed rate of protein synthesis is partially depend- ent 011 the sperific activity of the isotope in t,he tissue. This effect has been discussed in det,ail by scvcral investigators (33-35).

4. The tnost convenient, measure of collagen synthesis. is the dctcrtnination of labeled hvdroxvnroline. The other maior lune: protein known to contain hydroxyproline is clastin (3-8). “Elastii contributes less than 3 to 4@i0 t.o a hydroxyproline determination in lung homogcnatcs since the ratio of collagen to clastin in lung is 2 to 4: 1 and the content of hydroxyprolinc in lung etastin varies from 1.2 to 2% of the total amino acid residues (3-8). If lung collagen contains the usual amount of hydroxyproline for collagens (8 to lo%), a lo-fold change in the rate of synthesis of hydroxyprolinc would require a 150.fold change in the total amount. of elastin if only clastin were contributing to the measurement of hydroxyproline.

RZSULTS I 1

Conlposition of Rabbit Lung Collagen-Alt~hough useful quan-

titics of collagen cau be extracted from human fetal lung (12 to

18 weeks of gestational age) with 0.5 M acetic acid (12), this pro-

cedure gave low yields with early adult (2 month) rabbit luug.

For this reason, we attempted to develop lathyrism iu young

rabbits to incrcasc the quantities of extractable lung collagen.

The doses used produced clinical evidences of lathyrism in the

rabbits, but the yield of extractable lung collagen was still low.

Even so, the dose of 0.3 7. (w/w) P-aminopropionitrile in con-

vcntional rabbit feed for 10 weeks did prove sufficient to allow

the extraction of enough noncross-linked lung collsgcn for analy-

sis.

Rabbit luug collagen can bc scparatcd into crt, (Ye, pl1, and

/3tz components on CXcellulose chromatography by conven-

tional tcchuiques (Fig. I A). The 011 aud 012 componeuts elute at

apprositnately the same ionic strength as do pig skin cyl and (Ye

chains (data not shown) or rabbit skin collagen chains (in Fig.

IB, the carrier collagen is frotn the skiu of lathyritic rabbits).

Each major peak was pooled as iudicatcd aud electrophoresed

011 sodium dodecyl sulfate gels aud acidic gels, each dernonstrat-

iug greater than 90% homogeneity. These preparations co-

electrophoresed witjb calf, pig, and rabbit skin aI, CQ, /3tI, and

/?12 components, verifying their identity. Molecular weights

estimated on sodium dodecyl sulfate gels are approsimately

100,000 atId 200,000 for the a and /I components, respectively.

The amino acid composition of rabbit lung Q, and 02 chains

from ucwborns aud adults is similar but uot identical (Table I).

Amino acid analysis of each of these chains is characteristic for

collagen, but is not specific for lung. Comparison of lung col-

lagen with the amino acid composition of rabbit skin Q, and LYZ

(37) shows near identity except that: (a) there is more hydroxy-

lysiiie iu lutig a1 and (Ye chains and (b) there is a small amount of

3-hydrosyproline prcscnt both in rabbit lung 011 and 01~ chains.

The hytlrosyproline to prolinc ratio in collagen chains extracted

from adult rabbit lungs is slightly higher than that found in col-


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TABLE I

Amino acid compositio>l of rabbit lung collagen and noncollagen protein al different ages

Two milligrams of each preparation were hydrolyzed in con- tained from lathyritic rabbits. The adult lung collagen a2 chain stant boiling HCl at 110” for 24 hours and chromatographed on a methionine data may be overestimated secondary to base-line

Beckman 120 B analyzer with a continuous gradient for the acidic changes in the amino acid analyzer during its determination, and neutral amino acids (30). The data are given as residues per The similarity of the average composition of adult lung 01~ and 01~

1000 total residues. A correction of 4(y0 was used for serinc hy- chains with ~12 and their relative molecular weights (Fig. 3) sug-

drolytic losses. There were no other significant differences in gest, that plz is a dimer of 011 and (~2. The collagen composition

24-, 4%, and 72-hour hydrolysates. The analyses on collagen LYI, data in the newborn group were obtained from normal rabbits.

(~2, &, and p12 components were done on proteins that appeared The noncollagen protein composition data were obtained from to be more than 90% pure on sodium dodecyl sulfate gels. The normal rabbits after the collagen had been removed (see “Ex- collagen composition data in the 4-month-old group were ob- perimental Procedure” for details).

Hydroxylysine

Lysine

Histidine

Arginine

4-Hydroxyproline

3-Hydroxyproline

Aspartic acid

Threonine

Serine

Glutamic

Proline

Glycine

Alanine

Half Cystine

Valine

Methionine

Isoleucine

Leucine

Tyrosine

Phenylalanine

r- Collagen

13.2 13.3

31.8 25.4

2.5 8.9

61.4 64.8

91.2 88.8

1.7 1.2

42.4 43.1

17.1 21.6

33.9 33.3

76.5 69.1

.12 96.9

137 832

-14 01

0 : 0.5

19.0 28.2

7.8 11.0

7.6 15.2

18.7 33.0

2.2 1.9

9.2 11.2

4-Month-Old

Bll

11.4

29.3

2.1

55.3

LOO

42.2

16.9

31.8

77.1

116

342

115

0

16.2

5.5

8.3

19.4

12.4

1

3

1

-

612

13.2

28.5

7.7

62.1

91.1

1.9

42.1

20.0

33.5

73.4

01

40

05

0

23.4

7.2

11.5

25.1

1.6!

10.9

lagen chains from early fetal human lung, adult rabbit skin, or

newborn human skin (Table II). The ratio of cyl to (Ye chains in lung collagen extracted with

0.5 M acetic acid from lungs of lathyritic adult rabbits is 2 : 1 as

estimated by CM-cellulose chromatography. On sodium dodecyl sulfate and acidic gels, under conditions in which rabbit skin and calf skin have a chain ratio of 2 : 1, adult rabbit lung also has a ratio of (Ye to o(2 chains of 2: 1.

Composition of Rabbit Lung Noncollagen Protein-Three con- secutive extractions of rabbit lung homogenate in 10% trichloro-

T Newborn

a1 + a: 0.1 a2

2 1 ,

13.2

28.6

5.7 34.71 30.11 46.9

63.1 52.41 53.81 43.6

90 <l <l <l

1.5 0 0 0

42.7 95.0 89.8 83.9 I I

19.4 50.7

33.6 62.7 65.3 62.1 I I

72.8 108 119 104

104 46.8 45.4 49.2

335 77.3 81.6 84.5

107 I1 85.9 90.0 97.0 I I

< 0.25 4.31 7.01 5.7

23.6 70.0 71.3 77.9

9.4 15.3 17.3 14.7

11.4 34.3 39.0 29.7

25.8 92.0 97.2 102

2.1 23.1 27.1 23.8

10.2 36.7 36.7 41.0

I I I I

acet.ic acid at 90” for 30 min removed essentially all the collagen as evidenced by the lack of hydrosyproline or hydrosylysine in the hydrolysate (Table I). The trace amounts of hydrosypro- lint (<I residue per 1000) wcrc probably due to the presence of elastin, which was not solubilized by this treatment (3-8). The relative amounts of each amino acid in lung noncollagen protein were the same in the newborn and adult lung (Table 1).

Identification of in Vitro Product-Lung tissue slices synthesized collagen as identified by charge (CM-cellulose chromatography (Fig. 1B) and acidic polyacrylamide gels (Fig. 4, B and C)),

7.8

38.6

5.4

53.5

91.1

1.1

48.3

19.7

39.1

80.4

08

15

09

1.5

24.3

8.2

9.4

21.8

3.8

13.9

9.7

23.6

7.9

52.4

84.2

0

45.2

20.9

38.9

73.4

103

345

04

0

28.6

5.2

13.2

27.9

2.4

14.4

Noncollagen

Protein


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TABLE II TABLE III

Comparison of hydroxyproline to proline ratios in collagen from several sources

The data from rabbit skin (37) and human skin (38) were taken

Identification of lung collagen by sensitivity to collagenase or to trichloroacetic acid at 90”

from the literature. The ratio of [3H]hydroxyproline to [3H]proline in (11 and (~2 chains separated on CM-cellulose was determined by hydrolyzing 5 X lo5 cpm of [3H]cr1 and [3H]~2 with carrier rabbit lung collagen in constant boiling HCl at 110” for 24 hours. Hy- droxyproline and proline were isolated on a Beckman 120 B amino acid analyzer, 2-ml fractions were collected, and each fraction was counted in 18 ml of Aquasol, with an efficiency of 25%. The [3H]hydroxyproline to [3H]proline ratio in homogenates from 2- hour incubations of rabbit lung slices from late fetal (minus lo- day-old) and 2-month-old rabbits are similar, further validating the use of labeled hydroxyproline as a measure of collagen synthesis at different ages. Likewise, the sum of the proline and hydroxyproline residues in lung collagen (~1 or a2 does not change with age nor does the sum of prolinc and hydroxyproline residues in lung noncollagen protein change with age (Table I). Thus, the formula given in the legend to Fig. 9 can be used with validity to compare th’e rates of collagen synthesis at different ages.

Approximately 8000 cpm of [3H]al and [3H]az obtained from CM- cellulose columns similar to Fig. 1B were incubated with a purified bacterial collagenase or 10% trichloroacetic acid at 90” as described in “Experimental Procedure.” After incubation, the remaining intact protein was precipitated with 10% trichloroacetic acid at 4’ for 10 min, collected on celluose nitrate filters, and washed with 5% trichloroacetic acid. The filters were dissolved in Bray’s scintillator and counted. These values were compared with parallel incubations with approximately 9000 cpm [3H]tryptophan-labeled noncollagen protein obtained from rabbit lung (see “Experimental Procedure”) or approximately 8000 cpm [3H]leucine-labeled globin obtained from rabbit reticulocytes (39). Only the labeled collagen is significantly sensitive to col-

lagenase or trichloroacetic acid at 90”.

Incubation conditions

Source

In vitro synthesized collagen Rabbit lung, late fetus.. Rabbit lung, 2 month.,

Extracted Collagen Rabbit lung, newborn. Rabbit lung, 4 month. Human lung, early fetus.. Rabbit skin, adult.. Human skin, newborn.

Ratio of hydroxyproline to praline

a~ Chain

0.72 0.70

0.72 0.72

0.85 0.82

0.83 0.93 0.79 0.74 0.78 0.82

0.68 0.69

a2 Chain

by molecular weight (sodium dodecyl sulfate acrylamide gels (Fig. 3)), by sensitivity to purified bacterial collagenase (Table III), by sensitivity to trichloroacetic acid at 90” (Table III), and by the incorporation of labeled proline into protein and its con-

version to labeled hydrosyproline (Table II). The [3H]a1 and [%]cuz chains co-chromntographed on C%cel-

lulose, not only with rabbit lung collagen chains, but also with pig skin or rabbit skin al and (Ye chains. Rabbit lung [%]a1 chains cochromatographed with human fetal lung (Ye chains, but rabbit lung [3H]crz chains eluted before human fetal lung a2 chains on CM-cellulose (12).

The (Y, to CQ ratio of the in vifro product from 2-month-old rabbits was 2: 1 (ratio derived from Fig. 1B after correction for the relative differences in the sum of proline and hydrosyproline between the two chains (Table I)).

In all C&I-cellulose chromatograms of the in vitro product, there was invariably a peak of radioactivity preceding the al chains. This radioactivity moved into the cyl region with longer incubation (“chase”) times after a 20.min “pulse” with [3H]proline in the incubation medium (Fig. 2). This peak is probably pro-a1 chains, a higher molecular weight precursor of o(1 that has been demonstrated in other tissues (40-43). The [3H]pro-Crl had an approximate molecular weight of 120,000 and it coelectro- phoresed on sodium dodecyl sulfate gels with pro-a1 chains isolated from dermatosparasic cows (Fig, 30).

Similar patterns of incorporation ca.n be demonstrated on

CM-cellulose with [3H]glycine or ITith [%]methionine. When [%]cystine was used as a label, however, there was incorporation into the pro-Lyl region, but none into the o(~ region (data not

Tricbloroacetic acid- precipitahle

[3H]Proline-ayl (rabbit lung). 7320 + Collagenase............................. 560 + Trichloroacetic acid, 90”. 280

[3H]Proline-cuz (rabbit lung). 7676 + Collagenase.............. .._......_. 852 + Trichloroacetic acid, 90”. 760

[%]Tryptophan-protein (rabbit lung). 8822 + Collagenase............................. 7806 + Trichloroacetic acid, 90”. 6714

[3H]Leucine-globin (rabbit reticulocyte) 7629 + Collagenase............................. 7893 + Trichloroacetic acid, 90”. 7083

shown). This result is consistent with the finding of no cystine in rabbit lung LYE chains and of the presence of cystine in the precursor segment of pro-al, as in other tissues (40, 41). We have not been able to recover enough unlabeled pro-al from rabbit lung to do composition studies, nor has a quantity of radioactive pro-a2 chains been isolated as yet.

On sodium dodecyl sulfate gels, the [“HIa and [3H]a2 chains co-clectrophoresed with carrier rabbit lung, human lung, calf skin, pig skin, or rabbit skin cyl and (~2 chains (Fig. 3). All of these chains have a molecular weight of approximately 100,000 (12,44). When the entire [3H]al and [3H]~2 fractions on CM-cellulose were isolated and elect,rophoresed on sodium dodecyl sulfate gels, there were very few higher molecular weight components present (Fig. 3, B and c). This result is expected, because of the presence of @aminopropionitrile in the incubations.

On acidic polyacrylamide gels, [3H]o(l and [W](Y~ chains synthesized in vitro co-electrophorescd with carrier (Ye and a2 chains from rabbit lung, rabbit skin, or human fetal lung (Fig. 4).

The purified bacterial collagenase used did not significantly hydrolyze noncollagen proteins ([3H]leucine-labeled globin or [31-I]tryptophan-labeled noncollagen lung protein) (Table III). After incubation with bacterial collagenase for I hour at 37”, 90% of the [3H]~1 and [3H]a2 chains synthesized in vitro were no longer precipitable with trichloroacetic acid (Table III).

More than 90% of the [%]a1 and [3H]crz chains isolated from C11-cellulose were hydrolyzed by 10% trichloroacetic acid at 90” and were no longer precipitable by trichloroacetic acid at 4” (Table III).

The ratio of [3H]hydroxyproline to [3H]proline in 01~ and LYE chains synthesized in vifro and isolated by CM-cellulose chromatography was similar to that found in extracted collagen


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FIG. 2. Carboxymethylcellulose chromatography of rabbit lung a chains synthesized at different incubat,ion times. Lung slices of 300 mg (wet weight) from 3- to 4-day-old rabbits were incubated: A, with [3H]proline for 20 min; B, with [3H]proline for 20 min, then with fresh medium with unlabeled proline for 20 min; C, wit.h (3Hlproline for 20 min, then with fresh medium with unlabeled proline for 1 hour. Counts per min per fraction (O- - -0) and absorbance at 230 nm (---) of rabbit lung collagen carrier are shown. The ratio of cpm in the peak of radioactivity (pro-a,) eluted before the a, carrier decrease with respect to the cpm of radioactivity (LYE) cochromatographing with the 011 carrier as the incubation time was increased. The variable peak of counts per min eluting before the pro-al peak was not hydrolyzed by collagenase.

chains from newborn or adult lungs (Table II). In addition, this ratio is the same at different stages of development.

Lung Coonqosition during Development-From fetal ( - 10 days) to adult life (3 years) the wet weight of the lung increased lOO- fold, whereas rabbit weight increased lOOO-fold. From -10 days to about 1 to 2 weeks after birth, the wet weight of the lung was 2 to 2.5% of total body weight. In the 3-month-old rabbit this percentage declined to 0.4yG, then remained constant (data not shown).

The concentration of DNA (related to dry weight) in rabbit lung was relatively higher in fetuses than in adults, but just after birth it stabilized at approximately 38 pg per mg (Fig. 5A). The concentration of hydroxyproline, however, rose dramatically from 2 pg per mg in fetal life (- 10 days) to 13 c(g per mg at 3 years (Fig. 5C). This represents an estimated change in the concentration of collagen from 15 to 100 I.rg of collagen per mg of dry weight during this time.

The total protein in the lung (Fig. 5B) was measured by the Lowry method, which is approximately half as sensitive for collagen as for albumin (32). I f we assume 7 to 8 pg collagen per pg of hydroxyproline (Table I), then the protein per dry weight curve would change slightly (approximately lo%, higher as the rabbit ages) when this correction is made.

Conditions for Determining Rates of Protein Synthesis-The specific activity of the [14C]proline used in the rate studies is

FIG. 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of rabbit lung collagen. Gels were electrophoresed for 6 to 12 hours at 8 ma per gel. After electrophoresis, the gels were stained with Coomassie blue (30 min, 23”) and destained by diffu- sion in 7.0% acetic acid, 5y0 methanol (16). Gels containing labeled collagen were sliced into l-mm sections and prepared for counting by the addition of 50 ~1 of 30% Hz02 and incubated in capped counting vials for 8 to 12 hours at 50” before the addition of a Triton X-NO-based scintillator (14). A, 50 micrograms of partially purified lung collagen ext.racted with 0.5 M acetic acid from the lungs of lathyritic 4-month-old rabbits. B, 5000 cpm of [3H]- proline-labeled (~1 chains from rabbit lung isolated by CM-ccllu- lose chromatography. The carrier collagen was 50 pg of rabbit al chains. C, 5000 cpm of [3H]proline-labeled a~ chains isolated from lung by CM-cellulose chromatography. The carrier collagen was 50 pg of rabbit (YP chains. There is only a small amount of labeled p components in B and C because 8-aminopropionitrile was present in the incubation medium. D, 5000 cpm of [3H]proline-labeled lung pro-al chains isolated from lung [from Fig. 2B]. The carrier collagen is 50 rg of pro-al and pro-at chains isolated from dermatosparaxic cows. E, 50 pg of partially purified collagen extracted with 0.5 M acetic acid from the skin of 4-month-old lathyritic rabbits. P, 50rg of partially purified collagen extracted with 0.5 M acetic acid from lungs of 12- to B-week-old human fetuses (12). The absorbance at 570 nm (--) measured with a Gilford gel scanner (0.1.mm slit, 0.5 cm per min) and the counts per min per slice (O- - -0) are shown. Estimations of molecular weight were made on parallel gels with myoglobin, ovalbumin, bovine serum albumin, phosphorylase a, 8-galactosidase, and thyroglobulin as st’andard markers.

diluted by the unlabeled proline in the tissue. Under conditions used in these studies (i.e. incubation medium without added proline and a 45-min incubation of the tissue slice before the addition of isotope to fresh medium) the following data were found.

1. The specific activity of the isotope in the tissue increased linearly as the concentration of the isotope in the medium increased (Fig. 6A).


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I 84, 01 E. I .5. H”rnO” FetDl Lung Collagen

1.0 -

05-

0 10 20 30 40 50 60 OISTANCE FROM TOP OF GEL him)

FIG. 4. Acidic polyacrylamide gel electrophoresis of rabbit lung collagen. Gels were electrophoresed for 3 hours at 2 ma per gel, stained with Amido schwarz, and destained by electrophoresis in 7% acetic acid (19). The gels were scanned and counted as described in the legend to Fig. 3. A, 50 rg of acid-extracted lung collagen from adult rabbits; B, 5000 cpm of [3H]proline-labeled 0(1 chains from rabbit lungs with carrier rabbit lung err; C, 5006 cpm of [“Hlproline-labeled CYZ chains from rabbit lungs with carrier LYE chains from rabbit lungs; D, 50 rg of acid-extracted skin collagen from adult rabbit; E, 50 pg of acid-extracted lung collagen from human fetuses. The material used in A through E was identical with that used in the corresponding material in Fig. 2. The absorbance at 570 nm (--) and the counts per min per slice are shown.

2. The specific activity of the isotope in the tissue was constant during the time of incubation (from 1 to 5 hours) and de- pended on the isotope concentration in the medium (Fig. 6B).

3. The synthesis of collagen (incorporation of [14C]proline into [i4C]hydroxyproline) showed about a 30-min lag before it was linear for more than 5 hours (with respect to time) (Fig. 7A). The rate of collagen synthesis was estimated by determining the level of (i4C)hydroxyproline in each tissue slice at each time point (usually from 1 to 4 hours) and expressing it on the basis of DNA content and specific activity of the [i4C]proline in each tissue slice. The best straight line was estimated with these values (four time points in duplicate per rabbit) by least squares analysis. I f the correlation coefficient of fit was <0.9400, the experi- ment was discarded. The slope of t!ie line gave the incorporation of [i4C]proline into [i4C]hydroxyproline in collagen in nmoles of [r4C]hydroxyproline per mg of DNA*hour. An identical analysis was used for the rate of noncollagen protein synthesis, which was expressed in nmoles of [i4C]proline per mg of DNA.hour (Fig. 7B).

4. In order to relate the rate of collagen synthesis to the specific activity of the isotope, it was necessary to demonstrate a

P \, 0.6 -

S ProtemlOry Weight \E Z- Fi 04

I 6

@ -

_ 0.2 -

III I I I I I I I 1 1111 I I -10 t 20 60 100 140 180 “ I 2 3

BIRTH DAYS YEARS AGE

FIG. 5. Changes in lung composition with growth. A, DNA per dry weight; B, protein per dry weight; C, hydroxyproline per dry weight. The hydroxyproline concentration represents collagen concentration. The data in this figure were generated from the lungs of 300 rabbits. The error estimates shown are 1 S.D. of the mean.

linear increase in the rate of synthesis with an increase in the specific activity of the isotope in the tissue; this result is demonstrated in Fig. 6C. These determinations are important since the specific activity of the isotope in the tissue changed with age (Fig. 8)) even though the concentration of isotope in the medium was identical (0.02 mM) in these experiments. This age-related change in specific activity is, in part, secondary to changes in the total amount of proline in the tissue with age (Fig. 8).

The use of P-aminopropionitrile did not significantly alter the rate of collagen or noncollagen synthesis (Table IV). The use of ascorbic acid was necessary as a cofactor for collagen hydroxylation (27, 28) ; (Y, cr-dipyridyl almost completely inhibited hydroxylation (45)) but only partially affected noncollagen protein synthesis.

Comparison of Rates of Collagen and Noncollagen Synthesis during Development-Whereas the concentration of collagen in lung increased with growth, the rate of collagen synthesis mark- edly decreased with the major changes being between - 10 days and 1 month (Fig. 9.4). By 2 months of age, rabbit lung synthesized collagen at the same rate as did 2- to 3-year-old rabbit lung. The drop in the rate of noncollagen protein synthesis was even more marked, with the decrease occurring late in gestation (Fig. 9B).

The calculated ratio of the rate of collagen synthesis to the rate of noncollagen synthesis (expressed as a percentage) was low at -10 days, peaked between birth and 4 weeks, then declined to the -10 day fetal level where it remained throughout life (Fig. 9C).

DISCUSSION

Composition-The identification of rabbit lung collagen and its separation into (pi and a2 chains are quite similar to the identification and separation of collagens from other tissues (44) ; CM-cellulose chromatography, sodium dodecyl sulfate acrylamide gels, and acidic acrylamide gels all proved useful. The


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/

30

[‘C

Oles 1 I( 10-3)

FIG. 6. The use of the specific activity of the isotope in the tissue to compare the rates of protein synthesis in the rabbit lung. All of the data shown here were determined from lung slices from l-day-old rabbits. Similar data were found at each age group and for noncollagen protein synthesis as well as for collagen synthesis. A, the specific activity of [14C]proline in the lung slices (counts per min per nmole) as a function of the millimolar concentration of labeled proline in the incubation medium. There was no proline in the incubation medium other than that contributed by the isotope. The specific activity of the isotope added to the medium was 260 mCi per mmole. The lung slices were preincubated for 45 min in medium without isotope before the addition of isotope to fresh medium. As the concentration of the isotope in the medium increased, the specific activity of the isotope in the tissue (measured at 2 hours of incubation time) also increased. B, the specific activity of [‘*C]proline in the lung slices (counts per min per nmole) as a function of the time of incubation. The lung slices were preincubated as described above, then the isotope was added. The specific activity of the isotope in the tissue did not change during 1,2,3, or 4 hours of incubation with the isotope in the media. This was true at each of three levels of isotope in the medium (0.012, 0.024, and 0.036 mM). C, the rate of collagen synthesis in l-day-old rabbit lung slices (counts per min of [‘“Cl- hydroxyproline per mg of DNA per hour) as a function of the specific activity of [14C]proline in the tissue (counts per min per nmole). As the specific activity in the tissue increased, there was a linear increase in the apparent rate of synthesis of collagen. For this reason, a,.11 rate data are expressed in relation to the specific activity of the isotope in the tissue.

composition of lung collagen is characteristic of collagens found elsewhere in the body. It is composed of (at least) two chains, CX~ and (Ye, each of an approximate molecular weight of 100,000. The amino acid content is not specific for lung and is similar to that of collagen from rabbit (37), pig (46), calf (47), and human (38) skin, as well as human fetal lung (12). The relatively high proportion of hydrosylysine in rabbit lung o(~ and 0~2 chains may represent potential sites of either cross-linking or carbohydrate association, or both (44). The small quantity of the unusual amino acid 3-hydroxyproline may represent the presence of basement membrane collagen in the lung (48). Although the ratio of 3-hydroxyproline to 4-hydroxyproline in basement membrane collagen is usually much higher (48)) the relative amount of basement membrane collagen compared with the total collagen in the lung is probably quite small, so this ratio would be mark- edly reduced.

The ratio of a1 to a2 chains extracted from adult rabbit lung

and separated by CM-cellulose chromatography is 2: 1. In lungs from younger rabbits and human fetuses the ratio is higher, suggesting the presence of a heterogenous mixture of tropocol- lagens of the forms (011)~ and (c&x~.

The extracted collagen from the lungs of adult rabbits made lathyritic with P-aminopropionitrile has large amounts of cross- linked @) components (Fig. 1). This is probably a reflection of the low rate of synthesis of new lung collagen in this age group.

,$&e&-The identification of a portion of the proteins syn-

FIG. 7’. The synthesis of collagen and noncollagen protein in lung slices. A, collagen synthesis measured by the incorporation of [*4C]proline into [14C]hydroxyproline in collagen expressed in relation to DNA content and the specific activity of the isotope in each tissue slice (nanomoles of [J4C]hydroxyproline per mg of DNA). Collagen synthesis was measured at W-, I-, 2-, 3-, 4-, and 5-hour intervals in lung slices from a-week-old rabbits. There was a lag of about 20 to 30 min in the incorporation followed by a linear increase in collagen synthesis for more than 5 hours. Each data point represents the mean of three individual slices. A least squares fit regression analysis (Sony ICC-2550W calculator, program SO310) was applied to the l- to 5-hour data giving the rate of collagen synthesis (the slope) as 2.81 nmoles of [‘*C]hydroxyproline per mg of DNA per hour. With a correlation coefficient of 0.9955, the coefficient of variation is less than 2%. B, noncollagen synthesis measured by the incorporation of [I%]- proline into noncollagen protein (see “Experimental Procedure” for details). These data were obtained from the same slices as A and the calculations were performed in a parallel fashion. Non- collagen protein synthesis also had a lag before a linear incorporation from 1 to more than 5 hours. The determination of the rate of noncollagen protein synthesis of 8.10 nmoles of [‘%]proline per mg of DNA per hour had a correlation coefficient of 0.9970 and a coefficient of variation of less than 2%.

0 ,-, , , / / / , , ,

-10 , 20 GO 100 140 IAO’W

BIRTH AGE (days) AGE (yews)

FIG. 8. Changes in the specific activity of the isotope in the tissue with age. A, soluble [“Clproline that was not incorporated into either [14C]prolyl-tRNA or protein measured in lung slices from each age group. Data are expressed per mg of dry weight of the slice. B, total proline that was not incorporated into either prolyl-tR.NA or protein measured in lung slices from each age group. Data is expressed per mg of dry weight of the slice. C, the specific activit.y of [l%]proline in the lung slices at each age group given as counts per min per nmole. The data in C were calculated by dividing the data in A by the data in B at each age. The error estimates are the standard deviations of the means representing slices from 200 rabbits.

thesized by lung tissue slices in vitro as collagen (Y~ and (~2 chains can bc made in several ways (see “Results”). The availability of techniques to examine the synthesis of specific collagen chains by lung may be useful, for the following reasons.

1. As little as 100 mg (wet weight) of lung can be used to examine the kinds of cx chains synthesized in vitro by lungs. This


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TABLE IV Incubation conditions for in vitro synthesis of rabbit lung

collagen and noncollagen protein The rate of synthesis of collagen apparently decreases in the

presence of 2.5 mM @,a-dipyridyl because of the inhibition of hydroxylation of proline which is used as a measure of collagen synthesis. The synthesis of noncollagen protein is reduced by 250/, under these conditions. These data were from lung slices from a-week-old rabbits.

Rate of synthesis

Incubation conditions Collagen with Noncollagen [“Clhydroxy-

proline protein with [W]proline

nmoleslmg DNA/hr

Complete (p-aminopropionitrile, ascorbic acid)

+ a,ol-Dipyridyl - @-Aminopropionitrile.. - Ascorbic acid.

2.50 8.20 0.28 6.00 2.40 8.30 1.10 8.25

may be particularly useful in studying collagen synthesis in biopsy specimens from human lungs.

2. The use of /3-aminopropionitrile in the incubation medium prevents cross-linking (Fig. 3, B and C), while not influencing the rate of synthesis of collagen or of noncollagen protein (Table IV). This technique ensures that the collagen chains being synthesized by the lung at that particular stage in development are examined. In comparison, the efficiency of extraction of collagen synthesized previously in tissues depends upon the conditions used for extraction and the relative amounts of cross-linking present. For example, in human fetal skin there is a genetically distinct a!1 chain that cannot be extracted by conventional techniques (49).

It is unlikely that the over-all mechanisms of collagen synthesis in lung are different from those in other tissues. For example, the higher molecular weight species of lung collagen crl has several of the characteristics of the pro-al chain described in other tissues (40-43), and studies by Lapiere et al. (50) have demonstrated the presence of significant quantities of procollagen peptidase in normal bovine lung.

Early studies of the synthesis of collagen in lung yielded data in terms of turnover (51), but only in viva synthesis was examined. Comparisons of the rates of synthesis of proteins in whole animals are fraught with assumptions (52, 53) and are impossible in human lung. The use of tissue slices also has several problems associated with it, but these can be minimized by controlling the conditions of the incubation (33-35). The conditions we used in these studies meet the criteria of minimal assumptions (see “Results”). Most importantly, lung collagen and noncollagen synthesis is linear for more than 5 hours and the rates of synthesis of lung proteins can be expressed in relation to the average specific activity of the isotope in the tissue. Similar approaches have been used to study protein synthesis in diaphragm (54), pancreas (55), brain (33, 34), kidney (56), oviduct (35), and parotid gland (57).

Since labeled proline was the only amino acid used in the comparison of the rates of protein synthesis during development, it is important that the sum of proline and hydroxyproline in lung collagen and noncollagen protein be the same at different ages, as is the ratio of hydroxyproline to proline in both the neW]ji synthesized and extracted collagen chains (Tables I and II).

Changes in Lung Composition and Protein Synthesis with Age- The weight of lung in proportion to the total weight of the animal is much higher in fetal and early life. The relative proportion

A Rote of Collagen Synthms

0

--==+I 60 B Rate ot Non-Collagen Proiem Synthesis

5 r;lti I za 40 I\

t

C % Collagen Synfhms 16

A

g ‘!!1--- lso’1 2 3

BIRTH AGE (days) ffiE bears)

FIG. 9. A comparison of the rates of collagen and noncollagen protein synthesis during growth of the lung. The rates of each were calculated from data such as that shown in Fig. 7 except that values for 35 hour and 5 hours were omitted. A, the rate of collagen synthesis (incorporation of [“Clproline into [Wlhydroxypro- line in collagen given as nanomoles of [“Clhydroxyproline per mg of DNA per hour) determined at different ages. Each data point represents the linear slope of the pooled incorporation data from several rabbits. In all cases, the correlation coefficient was >0.9400 and the coefficient of variation was less than 5%. B, the rate of noncollagen synthesis (incorporation of [‘%]proline per mg of DNA per hour) determined at different ages. The statistical methods and criteria were the same as used in A. C, the calculated percentage of collagen synthesis measuring the relative proportion of the protein synthesizing machinery synthesizing collagen in lung tissue slices at different ages. The rates of incorporation of [‘4C]proline into noncollagen protein was mult,iplied by 4.13, which is the relative number of residues of proline and hydroxyproline in lung collagen compared with the number of residues of proline in lung noncollagen protein. The percentage of collagen synthesis was calculated at each age from the ratio of [rate of collagen synthesis X 1001 to [(rate of noncollagen protein synthesis) X 4.13 + (rate of collagen synthesis)]. The data shown in A through C were accumulated from a total of 300 rabbits.

(per gram of dry weight) of DNA in lung is slightly higher in the fetus, but this value levels off at about birth, probably reflecting a stabilization of the relative amount of extracellular mass by this time. Over-all protein concentration in the lung does not change significantly yet there is a marked increase (over 5-fold) in lung collagen (per unit weight of lung) from fetal to adult life. From early adult life on, however, the collagen concentration does not change significantly. This result is consistent with the finding by several investigators that the level of collagen in the human adult does not change with age (3-11).

The rate of collagen synthesis is high in late gestation and falls off gradually to a constant level by early adult life. The rate of noncollagen protein synthesis falls off even more rapidly, so that by birth, the level is essentially the same as in adult life. This results in the calculated percentage of collagen synthesis peaking between late fetal and the first month of life in rabbits. It is not


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known at this time whether these changes in lung collagen synthesis represent changes in over-all collagen synthesis or are isolated to specific anatomical regions such as the bronchial tree or vasculature. For example, Wolinsky (58, 59) has demonstrated changes in collagen synthesis in the aorta in hypertension and aging.

During the time the lung establishes itself as a gas-exchanging organ, there arc several changes in lung function (1, 2). The apparent change in gene expression noted here may reflect a change in the over-all emphasis of the protein synthesizing machinery toward collagen synthesis and then back to noncollagen synthesis. This may reflect the presence of elaborate con- trol mechanisms on the replicative, transcriptional, translational, and/or post-translational levels. These mechanisms may be part,icularly important in regard to the large group of human diseases associated with pulmonary fibrosis in which the lung presumably synthesizes an escess of collagen or synthcsizcs collagen in 11ew regions of the lung resulting in an inability of the lung to function normally in gas exchange (60). The data and techniques described here are being used in our laboratory to examine the heterogeneity of collagen synthesized in the early stages of development, to utilize a cell-free collagen synthesizing system to examine possible transcriptional and translation COII-

trol mechanisms of gene expression, and to categorize the human pulmonary fibrotic disorders 011 the basis of the types of collagen synthcsizcd.

Acknowledgments--We would like to thank Drs. W. F. Ander- ~011, G. Martin, R. Shulman, II. Keiser, J. l’icrcc, J. Collins, R/I. Cowan, N. Elson, and J. Last for helpful discussions and Ms. Diana Winters for editing the manuscript. l)rs. R. Shulman and J. Gardner kindly msdc amino acid analyzers available to us. Skin from lathyritic pigs was a gift from I>r. J. Pierce; 3-hy- drosyprolinc was a gift from Dr. F. Irreverrc; dcrmatosparasic bovine skin pro-crl and pro-Lyz were D <rifts from Dr. L. Kahn.

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Lung Collagen Composition and Synthesis: CHARACTERIZATION AND

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Lung Collagen Composition and SynthesisIn the lung, collagen is found associated with bronchi, with...

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